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Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

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Related in: MedlinePlus

LdAS-LdPEX5 interactions.(A) SDS gel of recombinant ASS proteins expressed in bacteria and purified over Ni-NTA resin beads, stained with Gel Code Blue (Upper panel). The lower panel corresponds to the anti-LdASS Western blot of the recombinant proteins. (B) Microtiter plates were coated with decreasing concentrations of recombinant LdASS forms or LdIMPDH and then incubated with LdPEX5. Bound LdPEX5 was quantified by an indirect ELISA using anti-LdPEX5 antiserum. Each assay was performed in triplicate and the average absorbance values were plotted as a function of the log of the PTS1 protein concentration using the ORIGIN 7.0 software. (C) Microtiter plates were coated with decreasing concentrations LdPEX5 and then incubated with PTS1 proteins. Bound PTS1 proteins were quantified by an indirect ELISA using anti-V5 or LdIMPDH antisera. (D) LdPEX5 pull-down assay: protein lysates from Ld1S2D were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody or preimmune sera (NRS) and the immuno-blot was bound with anti-LdASS antibody. (E) Nickel agarose pull-down (PD): Left panel: protein lysates from V5His-LdASSWT were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody (lane 1) or preimmune sera (NRS) (lane 2) or for nickel agarose PD (lane 3) and the immuno-blot was bound with anti-LdASS antibody. Right panel: immuno-blot using cleared lysates (lane 4) or nickel agarose PD proteins (lane 5) was bound with anti-LdPEX5 antibody.
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pntd-0001849-g004: LdAS-LdPEX5 interactions.(A) SDS gel of recombinant ASS proteins expressed in bacteria and purified over Ni-NTA resin beads, stained with Gel Code Blue (Upper panel). The lower panel corresponds to the anti-LdASS Western blot of the recombinant proteins. (B) Microtiter plates were coated with decreasing concentrations of recombinant LdASS forms or LdIMPDH and then incubated with LdPEX5. Bound LdPEX5 was quantified by an indirect ELISA using anti-LdPEX5 antiserum. Each assay was performed in triplicate and the average absorbance values were plotted as a function of the log of the PTS1 protein concentration using the ORIGIN 7.0 software. (C) Microtiter plates were coated with decreasing concentrations LdPEX5 and then incubated with PTS1 proteins. Bound PTS1 proteins were quantified by an indirect ELISA using anti-V5 or LdIMPDH antisera. (D) LdPEX5 pull-down assay: protein lysates from Ld1S2D were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody or preimmune sera (NRS) and the immuno-blot was bound with anti-LdASS antibody. (E) Nickel agarose pull-down (PD): Left panel: protein lysates from V5His-LdASSWT were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody (lane 1) or preimmune sera (NRS) (lane 2) or for nickel agarose PD (lane 3) and the immuno-blot was bound with anti-LdASS antibody. Right panel: immuno-blot using cleared lysates (lane 4) or nickel agarose PD proteins (lane 5) was bound with anti-LdPEX5 antibody.

Mentions: To analyze the interactions in sorting and trafficking, we first analyzed the LdPEX5-LdASS interaction and second we examined the potential colocalization of LdASS with LdPEX14. To this end, we used a bacterial expression system to produce the recombinant LdASS proteins in 4 different forms: the same coding sequences expressed in Leishmania described above (Fig. 1A) were ligated into bacterial expression constructs. Recombinant LdASSWT and LdASSG128S proteins with N- or C-terminal tags were purified using Ni2+-NTA agarose affinity matrix. SDS-PAGE analysis showed the correct sizes (49 kDa) of all of the purified recombinant epitope tagged LdASS proteins (Fig. 4A, upper panel) that reacted with anti-LdASS antibody (Fig. 4A, lower panel). The purified proteins were then used to assess the capacity of the glycosomal PTS1 targeting receptor protein LdPEX5 to recognize the PTS1 tripeptide signal Ser-Ser-Leu on the LdASS proteins using a modified enzyme linked immunoadsorbant assay (ELISA) [39]. In the first experiment, microtiter plates were coated with the three variants of the epitope tagged LdASS and the LdIMPDH, a glycosomal enzyme that contain the C-terminal tripeptide PTS1 targeting signal Ala-Lys-Met, as positive control [37]. The data showed that LdPEX5 bound LdIMPDH and the N-terminal tagged LdASS proteins (both wild type and mutant) with comparable affinities that had a apparent dissociation constant (Kd) of ∼5 nM (Fig. 4B) which agrees closely with binding constants reported for LdPEX5 [39]. In contrast, no significant LdPEX5 binding was detected with the proteins (both wild type and mutant) in which the C-terminal PTS1 signal sequence was modified by the addition of epitope tags (Fig. 4B, data for rLdASSG128S-V5His, not shown). Comparable nanomolar binding affinities were obtained for the LdPEX5-V5His-LdASS and LdPEX5-LdIMPDH interactions using a constant amount of LdPEX5 immobilized on the microtiter plates and varying the level of V5His-LdASS or LdIMPDH in the binding mixture (Fig. 4C). Again no LdASS-V5His binding to LdPEX5 was detected. These experiments show that the tripeptide Ser-Ser-Leu is a bona fide PTS1 signal sequence recognized with high affinity by LdPEX5. Moreover, these experiments confirm that the Ser-Ser-Leu sequence must be located at the C-terminus of the protein to be bound the PTS1 receptor.


Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

LdAS-LdPEX5 interactions.(A) SDS gel of recombinant ASS proteins expressed in bacteria and purified over Ni-NTA resin beads, stained with Gel Code Blue (Upper panel). The lower panel corresponds to the anti-LdASS Western blot of the recombinant proteins. (B) Microtiter plates were coated with decreasing concentrations of recombinant LdASS forms or LdIMPDH and then incubated with LdPEX5. Bound LdPEX5 was quantified by an indirect ELISA using anti-LdPEX5 antiserum. Each assay was performed in triplicate and the average absorbance values were plotted as a function of the log of the PTS1 protein concentration using the ORIGIN 7.0 software. (C) Microtiter plates were coated with decreasing concentrations LdPEX5 and then incubated with PTS1 proteins. Bound PTS1 proteins were quantified by an indirect ELISA using anti-V5 or LdIMPDH antisera. (D) LdPEX5 pull-down assay: protein lysates from Ld1S2D were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody or preimmune sera (NRS) and the immuno-blot was bound with anti-LdASS antibody. (E) Nickel agarose pull-down (PD): Left panel: protein lysates from V5His-LdASSWT were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody (lane 1) or preimmune sera (NRS) (lane 2) or for nickel agarose PD (lane 3) and the immuno-blot was bound with anti-LdASS antibody. Right panel: immuno-blot using cleared lysates (lane 4) or nickel agarose PD proteins (lane 5) was bound with anti-LdPEX5 antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475689&req=5

pntd-0001849-g004: LdAS-LdPEX5 interactions.(A) SDS gel of recombinant ASS proteins expressed in bacteria and purified over Ni-NTA resin beads, stained with Gel Code Blue (Upper panel). The lower panel corresponds to the anti-LdASS Western blot of the recombinant proteins. (B) Microtiter plates were coated with decreasing concentrations of recombinant LdASS forms or LdIMPDH and then incubated with LdPEX5. Bound LdPEX5 was quantified by an indirect ELISA using anti-LdPEX5 antiserum. Each assay was performed in triplicate and the average absorbance values were plotted as a function of the log of the PTS1 protein concentration using the ORIGIN 7.0 software. (C) Microtiter plates were coated with decreasing concentrations LdPEX5 and then incubated with PTS1 proteins. Bound PTS1 proteins were quantified by an indirect ELISA using anti-V5 or LdIMPDH antisera. (D) LdPEX5 pull-down assay: protein lysates from Ld1S2D were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody or preimmune sera (NRS) and the immuno-blot was bound with anti-LdASS antibody. (E) Nickel agarose pull-down (PD): Left panel: protein lysates from V5His-LdASSWT were used in a co-immunoprecipitation reaction with Anti-LdPEX5 antibody (lane 1) or preimmune sera (NRS) (lane 2) or for nickel agarose PD (lane 3) and the immuno-blot was bound with anti-LdASS antibody. Right panel: immuno-blot using cleared lysates (lane 4) or nickel agarose PD proteins (lane 5) was bound with anti-LdPEX5 antibody.
Mentions: To analyze the interactions in sorting and trafficking, we first analyzed the LdPEX5-LdASS interaction and second we examined the potential colocalization of LdASS with LdPEX14. To this end, we used a bacterial expression system to produce the recombinant LdASS proteins in 4 different forms: the same coding sequences expressed in Leishmania described above (Fig. 1A) were ligated into bacterial expression constructs. Recombinant LdASSWT and LdASSG128S proteins with N- or C-terminal tags were purified using Ni2+-NTA agarose affinity matrix. SDS-PAGE analysis showed the correct sizes (49 kDa) of all of the purified recombinant epitope tagged LdASS proteins (Fig. 4A, upper panel) that reacted with anti-LdASS antibody (Fig. 4A, lower panel). The purified proteins were then used to assess the capacity of the glycosomal PTS1 targeting receptor protein LdPEX5 to recognize the PTS1 tripeptide signal Ser-Ser-Leu on the LdASS proteins using a modified enzyme linked immunoadsorbant assay (ELISA) [39]. In the first experiment, microtiter plates were coated with the three variants of the epitope tagged LdASS and the LdIMPDH, a glycosomal enzyme that contain the C-terminal tripeptide PTS1 targeting signal Ala-Lys-Met, as positive control [37]. The data showed that LdPEX5 bound LdIMPDH and the N-terminal tagged LdASS proteins (both wild type and mutant) with comparable affinities that had a apparent dissociation constant (Kd) of ∼5 nM (Fig. 4B) which agrees closely with binding constants reported for LdPEX5 [39]. In contrast, no significant LdPEX5 binding was detected with the proteins (both wild type and mutant) in which the C-terminal PTS1 signal sequence was modified by the addition of epitope tags (Fig. 4B, data for rLdASSG128S-V5His, not shown). Comparable nanomolar binding affinities were obtained for the LdPEX5-V5His-LdASS and LdPEX5-LdIMPDH interactions using a constant amount of LdPEX5 immobilized on the microtiter plates and varying the level of V5His-LdASS or LdIMPDH in the binding mixture (Fig. 4C). Again no LdASS-V5His binding to LdPEX5 was detected. These experiments show that the tripeptide Ser-Ser-Leu is a bona fide PTS1 signal sequence recognized with high affinity by LdPEX5. Moreover, these experiments confirm that the Ser-Ser-Leu sequence must be located at the C-terminus of the protein to be bound the PTS1 receptor.

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Show MeSH
Related in: MedlinePlus